Project Summary The long-term goal of the PI is to develop highly specific, RNA-targeted, small molecule ligands to probe the dynamic structure, fundamental biology, and therapeutic potential of long noncoding RNAs (lncRNAs). Select lncRNAs have been found to play critical roles in cancer progression, including lncRNA HOTAIR, which is implicated in metastatic breast cancer, and lncRNA MALAT-1, which is over-expressed in several cancers, including non-small cell lung cancer. Despite the proposed therapeutic potential of lncRNAs, adequate small molecule targeting strategies have yet to be realized. This slow progress is due in part to the nature of RNA as a dynamic structure with limited chemical functionality but also to a gap in knowledge with respect to guiding principles and methods for small molecule:RNA interactions. Our central hypothesis is that the parallel discovery of small molecule chemical space and RNA topological space privileged for differentiation will yield fundamental insights into small molecule:RNA recognition that can be applied to the rapid development of ligands with high affinity and specificity for a wide range of RNA targets. In prior work, the PI has identified common chemical properties of biologically active RNA ligands, elaborated RNA binding scaffolds for improved recognition of small RNA targets, revealed RNA secondary structures that can be differentially recognized by small molecules, and computationally identified ?hot spots? for targeting lncRNA HOTAIR. In the proposed work, we will simultaneously pursue two independent but complementary lines of fundamental investigation and apply the developed guiding principles and technologies to two critical lncRNA targets. In Area 1, we will use cheminformatic analysis, organic synthesis, and rapid screening methods to identify small molecule properties biased toward specific RNA recognition. In Area 2, we will use pattern recognition protocols to identify RNA structures that are readily differentiated by small molecules. In Area 3, we will combine our RNA- biased libraries and optimized screening assays to identify the first inhibitors of lncRNA tertiary structure, particularly the 3'-triple helix of MALAT1. In Area 4, we will use a wide range of computational and experimental tools to identify small molecules that inhibit lncRNA:protein interactions, viz. HOTAIR and its protein binding partner, PRC2. The rationale for this research is that our novel RNA-specific libraries and technologies will enable new investigations of RNA structure and function and serve as a rich platform for future development of RNA targeted therapeutics. In summary, our work will (i) produce the first widely available RNA-biased molecular library with demonstrated affinity and specificity for RNA targets; (ii) develop a range of computational, synthetic, and screening tools to enhance RNA ligand identification; and (iii) yield first- in-class technologies to develop small molecule ligands for lncRNAs. These distinct but complementary approaches will not only ensure success of this research and progress in the field of small molecule:RNA targeting, but will also provide lead small molecules for a range of critically important targets in human health.